Vsmcs

VSMCs were purchased from ATCC (Manassas, VA, USA). Cells were incubated in McCoy's 5A medium (Sigma) supplemented with 10% foetal bovine serum (FBS, Sigma) in 5% CO₂ at 37°C. ox‐LDL (Sigma) was utilized to make an aberrant lipid environment.

Vascular smooth muscle cells (VSMCs) (ATCC, Manassas, VA) were cultured in DMEM containing 10% bovine serum (Gibco, Grand Island, NY) under 37℃ and 5% CO2 condition. In the Ang II group, cells were incubated with 1.0 μM Ang II (Sigma-Aldrich, St. Louis, MO) for 24 hours. For intervention study, 40 μM MKC-3946 (Selleck, CA) were added before Ang II treatment.

Human vascular smooth muscle cells (VSMCs) were purchased from ATCC and cells were cultured in Dulbecco's Modification of Eagle’s Medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin in a humidified 5% CO₂ incubator at 37 °C. Cells were used between passages 3 and 6 for all experiments and the medium was changed every other day.
After reaching 80% confluence, VSMCs were incubated for 12 days in medium supplemented with 10 mM β-GP (G9422, Sigma, Silicon Valley, USA). For some experiments, VSMCs calcification was induced with 10 mM β‐GP after knockdown of RTEF-1 by small interfering RNA (siRNA) transfection or overexpression of RTEF-1 by stable trans-infectants. For manipulation, the activity of WNT/β-catenin signaling, LiCl (5 mmol/L), or Dickkopf-1 (Dkk1) (100 ng/ml) was added to the medium, as indicated in figure legends.

Human vascular smooth muscle cells (VSMCs; ATCC, USA) were cultured in Dulbecco’s
Modified Eagle Medium (DMEM) with 10% FBS and incubated in 5% CO₂ at
37 ℃. miR-34a mimics/inhibitor or negative control were transfected into VSMCs
after seeding 24 h in 6-well plate. The concentration of all RNAs used for
transfection was 50 nM based on the protocol of Lipofectamine 2000 (Invitrogen,
USA). For CXCR4 overexpression plasmid transfection, the concentration of
plasmid was 2 μg/well in a 6-well plate after 24 h of seeding. All analysis were
conducted after 48 h transfection.

Human vascular smooth muscle cells (VSMCs) were purchased from American Type Culture Collection (Manassas, VA, USA). VSMCs were cultured in F12 medium (HyClone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah, USA), 100 U/mL of penicillin-streptomycin and insulin-transferrin-selenium (Gibco, Thermo Fisher Scientific, Eugene, Oregon, USA). After VSMCs were grown to 70-80% confluence, the cells were cultured with serum-free F12 medium for 24 h, and the conditioned medium was collected and centrifuged for 10 min at 1000 r/min. The supernatants were collected and filtered through a 0.22-μm pore size filter and were used as the VSMC-conditioned medium[31 (link)]. SCAP-overexpressing VSMC-conditioned medium was collected from SCAP-overexpressing VSMCs after 4 h of treatment with or without 20 μM lycorine. Human umbilical vein endothelial cells (HUVECs) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone, Logan, Utah, USA) supplemented with 10% fetal bovine serum and 100 U/mL of penicillin-streptomycin. HUVECs were seeded at a density of 1×10⁶ cells/mL. After the cells were grown to confluence and became quiescent, they were incubated with VSMC-conditioned medium and 100 μg/ml of LDL for 24 h..